The derivation of human embryonic stem cells (hESCs) has opened new avenues for studies on human development and it also provides a potential source of cells for replacement therapy. For example, the ability to genetically alter hESCs offers unique opportunities to study the mechanisms regulating lineage commitment during human development, establish new approaches to identify and screen drugs, and develop in vitro models of human disease. The feasibility of this approach is dependent on the identification of a locus in the genome that is easy to access through targeting and one that will be permissive to expression of the introduced genetic material in the undifferentiated ES cells as well as in a broad range of differentiated cell types generated from these cells. A general review of this approach is provided in Yates et al., Gene Therapy (2006) vol. 13: 1431-1439.
Previous studies aimed at expressing genes in hESCs and derivative lineages have used either lentiviral vectors or transgenes that integrate randomly into the genome. These approaches are problematic for a number of reasons, e.g., a randomly integrated vector can activate or suppress expression of endogenous genes through insertional mutagenesis, the vectors are often present in multiple copies, and their expression is subject to silencing.
Homologous recombination in mouse embryonic stem cells has been used to produce mice carrying a single copy of the transgene integrated into a predetermined site of the genome (see e.g., Shaw-White et al., Transgenic Res.; (1):1-13 (1993); Bronson et al., Proc. Natl. Acad. Sci. USA, 93(17:9067-72 (1996); Hatada et al., J. Biol., Chem., 274(2):948-55 (1999); Tang et al., Genesis, 32(3):199-202 (2002)). In these studies, the ubiquitous Hprt locus was used with limited and unpredictable success. It would be desirable to define an autosomal locus that allows strong and predictable expression of transgenes inserted through homologous recombination, but is difficult to identify chromosomal loci that fulfill these criteria. Exogenous transgenes may not harbor all of the sequences necessary and sufficient for proper regulation of transcription and may therefore be influenced by cis-regulatory elements near the site of insertion.
In the mouse, a locus known as Rosa26 locus meets these criteria because it is expressed in ES cells and many derivative tissues both in vitro and in vivo and new genetic material can be easily introduced into it through homologous recombination. WO 99/53017 describes a process for making transgenic animals that ubiquitously express a heterologous gene, wherein the heterologous gene is under the control of a ubiquitously expressed endogenous promoter, e.g., that of the mouse Rosa26 locus. R. Dacquin et al., Dev. Dynamics 224:245-251 (2002) and K. A. Moses et al., Genesis 31:176-180 (2001) utilize the transgenic mouse strain R26R obtained according to WO 99/53017 for the expression of heterologous genes. WO 02/098217 describes a method of targeting promoter-less selection cassettes into transcriptionally active loci, such as the Rosa26 locus. WO 03/020743 describes the expression of transgenes in vivo by targeting protected transgene cassettes into predetermined loci (e.g. the Rosa26 locus), such that the introduced tissue specific exogenous promoter has at least some tissue specific activity.
US 2006/0205077 describes a method for targeted transgenesis using the mRosa26 locus. U.S. Pat. No. 6,461,864 also describes the use of the mRosa26 locus in the production of genetically engineered non-human animals that express a heterologous DNA segment.